Our studies of various virologic and immunopathologic processes that occur when viruses and parasites replicate in the ocular microenvironment comprise five areas: (1) virus induced retinal degenerative processes; (2) the possible roles of viruses in human diseases; (3) molecular diagnosis and pathogenesis of cytomegalovirus (CMV) infections in man; (4) Varicella - zoster virus (VZV) infections of the eye and (5) Toxoplasma gondii infections of the retina. We have established a model system for studying retinal degenerative diseases, experimental coronavirus retinopathy (ECR). The virus is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of infectious virus and progressive retinal degeneration. This is the first retinal model to demonstrate a virus induced degeneration, viral persistence, a genetic predisposition to virus induced tissue damage and a virus triggered autoimmune response. Our goal is to determine the pathophysiological mechanisms and to identify genes involved in the retinal degenerative disease. During the past year we have evaluated the role of cytokines. Analysis of cytokine levels within the eye, revealed that IL-1, Il-6, TNF-a and IFN-g were elevated in ECR. IFN-g and granule mediated cytotoxicity by cytotoxic lymphocytes (CL) play a critical role in limiting viral spread and may contribute of immunopathology. CL were analyzed by immunocytochemical staining for CD8 T cells and by RT-PCR assays for the CL cytotoxic granule, granzyme B. Sequenctial anaylsis of IFN-g and granzyme B gene expression within the retina demonstrated that these genes are up-regulated at the time of clearance of infectious virus from the retina. These studies indicate that cytotoxic T cells and T cell products (IFN-g and granzyme B) and present within the retina and contribute to retinal viral clearance.Human CMV is a herpesvirus that is a major cause of blindness in children born with congenital infections and in immunocompromised individuals. It is difficult to study CMV latency in man. Therefore cell culture models of CMV replication and latency may provide insight into a rationale for alternative treatment modalities. In order to understand the retinal tissue tropism for CMV, we have extended our original studies of CMV replication in HRPE. The data indicate that regulation of HCMV infection of HRPE cells differed from that of human fibroblasts at both the levels of virus entry and transcription of the viral genome. These differences in CMV - host cell interactions in HRPE cells which may be instrumental in CMV activation, replication and spread within the eye. In a separate series of studies we have evaluated methods of diagnosis and treatment of CMV infections. These studies demonstrate the utility of this CMV RPE cell model system to evaluate virus replication and efficacy of antiviral therapy with antisense oligonucleotides. Human VZV infections of the eye can result in keratitis, uveitis, acute retinal necrosis or progressive outer retinal necrosis. However, a small animal model that replicates these diseases does not yet exist. We have shown that intravitreal inoculation of guinea pigs with VZV results in a chronic uveitis consisting of a mononuclear cell infiltrate in the posterior segment of the eye. We have utilized this model to evaluate VZV vaccines and found that immunization with VZV gE and gI recombinant proteins induced potent humoral and cellular responses that accelerated the clearance of VZV DNA and may neutralize virus within the eye. Studies are continuing to establish an in vivo modelfor VZV keratitis. We have developed molecular diagnostic methods using PCR analysis to detect herpesviruses. These assays are being used to distinguish between infectious and immunopathogenic posterior segment intraocular inflammation.